Regulation of cytosine methylation in ribosomal DNA and nucleolus organizer expression in wheat

.J. Mol. Riol. (1988) 204, 523-534

Regulation of Cytosine Methylation in Ribosomal DNA and Nucleolus Organizer Expression in Wheat R. B. Flavell?, M. O’Dell Department of Molecular Genetics A FRC Institute of Plant Science Research (Cambridge Laboratory), Mark Lane, Trumpington Cambridge CB2 2LQ, U.K.

and W. F. ThompsonS Department of Plant Biology Carnegie Institute of Washington 290 Panama Street, Stanford, CA 94305, U.S.A. (Received 8 January

1988, and in revised form 2 June 1988)

has been studied in wheat rRNA genes at nucleolar organizers Cytosine methylation displaying different activities. The methylation pattern within a specific multigene locus is influenced by the number and type of rRNA genes in other rDNA loci in the cell. One CCGG site 164 base-pairs upstream from the start of transcription is preferentially unmethvlated in some genes. Dominant, very active loci have a higher proportion of rRNA genes with unmethylated cytosine residues in comparison with recessive and inactive loci. It is concluded that cytosine methylation in rDNA is regulated and that the methylation patt,ern correlates with the transcription potential of an rRNA gene.

1. Introduction Plant, DNAs generally have a high content of 5 methylcyt’osine. This base may account for over 30 ‘jb of the cytosine residues. Most of the :i-methylcytosines are found in CpG dinucleotides or CXG trinucleotides (Wagner & Capesius, 1981; (iruenbaum et al., 1981). There is now a substantial body of evidence, for animal genes and animal viruses. suggesting that gene expression is often of specific associated with undermethylation cvtosinr residues, although several apparent exceptions to t,his picture have been described (Yisraeli & Szyf? 1984; Razin &. Riggs, 1980; Bird, 1984, 1986). In this paper we examine cytosine methylation in the rRNA genes of different nucleolar organizers of wheat to investigate: (1) if active genes are methylat,ed t.o an extent or in a pattern different from that of inactive genes, and (2) if methylation t Present address: John Tnnes Institute. Colney Lane, Norwich. Norfolk NR4 7UH, C.K. $ Present address: Department of Botany, North (‘arolina State T!nivrrsity, Raleigh. Xic 27695. C1.S.A.

changes when the activit,y of an rDNA locus is changed. In investigations int,o rDNA mrthylat,ion in mice, Rird et al. (1981) found that’ most of the rRNA genes lacked methylated eytosines at, the sites examined, but some were heavily methylated at most CpG sites. These latter genes were considered t,o be inactive because they resided in a chromatin conformation resistant to DNase T. In Xenopus the rDNA contains many methylated cytosine residues but a small region in the intergenic spacer is hypomethylated (La Volpe et al., 1983). However, this hypomethylation is not invariably correlated with activity. In plants, t’here have been many reports that’ rRNA genes contain methylated CpG residues (e.g. Uchimiya et nl., 1982: Steele-Scott et al., 1984; Ellis et nb., 1983; Gerlach & Redbrook, 1979). This observation is not’ surprising because between 18 and 30”/ of all the cytosine residues are methylated in plants (Wagner 8: (:apesius, 1981: Gruenbaum et al.. 1981). However. in more detailed st,udies on rDNA methylation in peas (Waterhouse et nl., 1986; Watson et nl., 1987) a CCGG site located near the promoter wa.s found to he preferentially

524

R. H. Flavell

Table 1 ,Vuclrolar

volumes

and compensation Sucleolar 113

“(‘hinew Spring” C’S with 613 deleted C’S with 1 B deleted

53?;-3 lOi&

in (‘8

volume in roots (pm3) 613 27+3 70*22

Results taken from Martini

wheat

51) 4.5 + - 0.35 25.5 * 0.5 9.5 * 0.3

& Flaw11 (1985).

unmethylated in a subset of the genes and the proport’ion of genes methylated atI this site changed during plant development. It, was suggested that the cyt’osine methylation levels may be related to rRNA gene expression. A similarly located site hypomethylated in a subset of the rRNA genes has been described in flax (Ellis et al.. 1983; Blundy rf al., 1987). The genes coding for the 18 S, 5.8 S and 25 S rRNAs are organized in tandem arrays at nucleolar organizers (NORs)t in all eukaryotes. In hexaploid wheat). the rRNA genes are localized at four NORs on chromosomes lB, IA, 51) and 6B (Longwell & Svihla, 1960; Flavell & O’Dell. 1976). In the variety “Chinese Spring” (CS) the number of rRNA genes at each NOR has been determined (Flavell & 0’l)ell. 1976) and the relative activity of each NOR has been estimated .b~ measuring the volume of each nucleolus (Mart,ml & Flavell, 1985). Tn CS root’ tip cells, the NOR on chromosome IB organizes a nucleolus that is approximately twice the volume of the nucleolus at the NOR on cahromosome 6B (Table 1). The nucleoli from the chromosome 51) and IA NORs are very small in this genotype. The greater activity of the chromosome 1B KOR over the chromosome 6B NOR is not due t,o a larger number of rRNA genes being clustered at the 1H locus. since t’he chromosome 6B rI)iYA locus possesses twice the number of rRXA genes present at the chromosome 1B rDNA locus (Flavell &, O’Dell, 1976). Thus, it is likely t,hat many genes are inactive at the 6B r-DNA locus. When chromosome 1H is deleted, the volumes of t,he 6B and 51) nucleoli increase in size to compensate (Table 1). This compensation presumably involves the act)ivation of rRNA genes. Similarly. when chromosome 6K is delet,ed, the volumes of the 113 and 51) nucleoli increase in size. When bhe number of chromosomes fB or 613 is increased in the cell, the volumes of individual 113 and 6K nucleoli decrease to caompensate. Thr decrease is considerably more dramatic when chromosome 1U from AegiEops umbrllulata is introduced into CS wheat (Martini et al., 1982). It can thus be inferred t,hat this chromosome carries a NOR that is dominant over t.hose of chromosotnes lB, 6R and 51) of wheat,. When t Abbreviations “(‘hinese Spring”:

used: NOR. nurleolar organizer: IO3 bases or base-pairs.

kb.

C’S,

et al.

present, the 1IT 1;OR presumably contributes mat~,~of the active rRXA genes. while suppressing the activity of the wheat rRNA genes. In other studies (Thompson k Flav~ll. 1988) these interpretations of the activities of different NORs in the different. genetic backgrounds haves been supported because NOR’ a,ctivity is rorrelatrd with the fraction of its rRjS.4 girds that ark hypersensit,ivc to DIVasr 1. In thr work dcsc*ribed hcrr. t ho penes at t hci different wheat NORIs displaying different a.(-tivit.irr have been distinguished in isolated DNA using restrictZion endonucleases. and their cBytjosinr methylation has been studied especially at (:( ‘( K‘ and (YXX motifs. The former motif is not c~lcavt~d by Hhal when the int’ernal (’ is tnt*thylatc~d. Methylat~ion of the latt>er motif was investigated by cornparing DNA restriction by ;Vsl~l and mptr I I. Both enz,vmt~s cleave (‘UX: sequences. but only JZspl cleaves if t’hr internal cytosine is met hylatcd. although JYspl does not cleave if tht t~xt~~rnal C’ ih met’hylat’ed, t’he frequency of this csytosinr tnet~hyla tion pattern in wheat is much Iess than that whc&rr t.hr int,ernal (’ is mrthylated ((:ruenbaum rf rrl.. 1981). The number of rRNA genes varies xubstantiall~ within and brt,ween plant species. If a species uses a fixed number of genes to satisfy its ribonomr requirements. then the number of cxwss itractivc genes might be variable between plants differing in tot,al rDN.4 content. We have used the opportunit! of having c&ely related wheat. genotypes differing by more than fourfold in total rl)!YA (*ontent to examine also how t.he cytosine methylation pat,tcsrtl in rI)NA varies with different) numbers of rRKA genes. The results show that man!’ rl)SA repeat units are tnet’hylat’ed a,t all sites examined, while others show heterogeneous cytosinr methylation pat terrrs. although &ain sites are preferentially unmet II>,lated. I~nmcthylatcd cyt’osincs arc’ not distributtld at random: t,heir distribution is related to the activity of the NOR in which they reside. The pattern of methylat,ion for a given locus is not fixed but varies with t.htl gchnetic backgrountl and t hc number and kind of rRNA genes within it. Th(s preferent’ially unmethylated residues in activr loci are localized in sequences that may play iIn import,ant role in regulating t,ranscbription.

2. Materials and Methods The variet\, . “(‘hinrsr Spring” ant1 thr ti~llown~ nullisomic (S)-~tetrasomicz (T) and tetrasomic~ derivatives of it uwe used: N;GHTBA. SIHTIA. TIH and T6H. In these the dosage of specific c~hromosomrs has ~WII manipulated. P.R. in h’6HTSA. the (iI (~hromosonws ha\ c’ been drlrt.rd and an additional pair of (iA &rotnosomw are present to c~ompwsatr (Sears. 19%). .4lso used wvre CS plants wnt~aining a pair of I IT ~~hror~10~of~~~~~from il. umhdlvlntcr (WP Martini czf /I/.. 1982) either in additiotl to the full C‘S c~hromosome complement (C’S+ I I’) or it1 place of c~hromosom~s 1B (C’S 1 I‘ (I IS)). IA (f’s 1 I7 (I ~1)) or 1D ((Y 1I’ ( I I))). The plants w~rc~ grown at about 20’-(’

Regulation

of Cytosine

Methylation

in continuous light until approximately 20 cm in height. The material was harvested and stored at -80°C. (a) DNA

blotting,

extraction

3. Results (a) Restriction endonuclease cleavage maps of the rRNA gene repeats Restriction endonuclease cleavage maps for some of the rRNA gene repeats of the variety “Chinese Spring” have been published by Appels & Dvorak (1982). From these and our own studies, involving cleavage of total DNA with different enzymes, Southern blotting and hybridization with cloned rDNA and sequencing of a cloned gene (Lassner & Dvorak, 1986; Barker et al., 1988), we have drawn the following conclusions, summarized in Figure 1. Restriction enzyme sites assayed within the regions specifying the mature ribosomal RNAs are identical between between all repeats, but there is variat’ion

endonuclease digestion, electrophoresis, hybridization

and autoradiography

The restriction enzyme digests were carried out in conditions recommended by Bethesda Research Laboratories. Of each DNA. 1 pg was digested with 5 units of enzyme for 16 h. Where digestions with HpaII and another enzyme were carried out, the HpaII digestion was done first. the salt concentration adjusted and then

:

TTH

525

the 2nd enzyme added. The digested DKAs were fractionated by electrophoresis in 1 y0 (w/v) agarose then transferred to a nitrocellulose filter by the procedure of Southern (1975). Each filter was baked at 80°C for 2 h after washing in 2 x SSC (SSC is 0.15 M-sodium chloride, 0.015 M-sodium citrate). Hybridization of the filters with 32P-labeled, nicktranslated, cloned rDNA (pTa71: Gerlach & Bedbrook. 1979) was carried out at 65°C in a hybridization buffer containing 0.6 M-sodium chloride, 10 mM-Pipes (pH 6.8), 1 mM-EDTA, 0.1 y0 SDS, 10 x Denhardt’s solution (2% (w/v) gelatin, 2% (w/v) Ficol 100, 204, (w/v) polyvinyl pyrolidine 360), 10 pg sonicated salmon sperm DNA/ml. for 16 h. The filters were then washed in 2 x SSC and 0.3 x SSC at 65°C to remove unbound DNA, dried and exposed to X-ray film at - 80 “C.

Frozen plant tissue was ground to a fine powder in a mortar, with liquid nitrogen and a small amount of sand. The powder was transferred to a 50 ml plastic tube containing 2 vol. extraction buffer (100 mM-Tris HCI (pH %O), 100 mM-NaCl, 50 mM-EDTA, 2% (w/v) SDS). Proteinase K (0.1 mg/ml) was then added to the extract, mixed and kept for 1 h at room temperature, with occasional mixing. After extraction with an equal volume of chloroform/phenol saturated with 0.1 M-Tris . HCl (pH 84), the mixture was centrifuged at 3000 revs/min for 10 min. The aqueous phase was removed and mixed with 2 vol. ethanol and the precipitated DNA removed with a spatula, transferred to a 10 ml plastic tube, air dried, then redissolved in 1 ml of 10 miv-Tris. HCI (pH 84), 1 mM-EDTA (TE) buffer. RNase A was added to 10 pg/ml and the solution incubated at 37°C for 1 h. ARer extraction with chloroform/phenol, the aqueous phase was removed and the DNA precipitated with ethanol. The DNA was redissolved in TE buffer.

(b) Restriction

in rDNA

TH I

T 6B

TH

0’

r

TH I

7 ,

<

:,

I:

T

v

’

H

7

I kb

1B

Figure 1. Restriction endonuclease maps for rDPu’A of CS wheat and A. umbellulata. The information to produce the maps was taken in part from Appels & Dvorak (1982), from the sequencing of one isolated gene (Barker et al.. 1988), and from genomir DNA, cleaved with restriction endonuclease, fractionated by electrophoresis and hybridized with rDNA. Xot all sites for a given enzyme are shown, only those relevant to the experiments described in this paper. lB, 6B and I L’ describe the chromosome carrying the rDNA. The only HpaII site (v ) shown is that which is preferentially nonmrthylated (see the text). Many other HpaII sites exist. T. 7’aqI; H, HinfI; D. DdeI; B. BamHI: E. EcoRT; bp. basrpairs.

H. H. Navel1 et al.

526

repeats in the intergenic spacer DNA. This variation is due to loss/gain of endonuclease recognition sites and to differences in the amount of DNA lying between Hinff and DdeI sites, which consists mainly of a tandemly repeated 135 bp sequence. The structures of the rRNA gene repeats at the 1B and 6B NORs were determined by studying the rDNA hybridization patterns in digests of DNAs extracted from plants with chromosomes 112 or 6R deleted in turn. The rRNA gene repeats on chromosome 6B are of two types, differing in overall length by approximately 135 bp (see Fig. 1). This length difference is localized on the ‘I’aql-7’aqI. Hinfl-Hi&, Ddel-DdeT or Hinff-11paTT fragments containing the intergenic spacer and is due to variation in the number of 135 bp repeat,s that occupy most of this DSA (Lassner & Dvorak, 1986). These two forms of the rDN4 repeat at the 6B KOR account for approximately 60(& of thp rDNA in “Chinese Spring” (Flavell & O’Dell. 1976). Most of the genes in the 1B NOR have a 7’aqT site deleted compared with the 6B rRSA genes, as shown in Figure 1. On the basis of EcoRI + HpalI and DdeT digests these 1K gene repeats have the same amount of intergenic spacer DNA as thcl shorter 6B rRNA gene repeats a.nd therefore arr likely to possess the same number of 135 bp repeats. In addition to these 1B NOR genes, which have also been described by Appels & Dvorak (1982), we have characterized a minor form that contains longer 7’aqI. Hinfl and Ddel spacer fragments and which therefore presumably cont,ain more 135 bp spacer repeats (see Fig. 8). These genes are localized on chromosome 1B because they are absent from lines lacking chromosome 1B but not from lines lacking chromosomes IA. 51) or 6H. Scans of aut,oradiograms reveal that these genes occupy less than 10’~~; of t’he total rDNA in euploid “(“hinesr Spring”. The rRSA genes on chromosome 51) have much shorter 7’aqI spacer fragments (Appels & Dvorak, 1982) but these genes occupy only a small percentage of the total rDNA and detailed maps of them have not) been published.

repeat

units, tot’al CS DNA was cleaved with HpaIT + EcoRT. NamHI, HyaIJ + HamHT, or EcoRI + IZhaT. fractionated by elect,roHlltl transferred to nitrocellulose phoresis. hybridized with 32P-labeled rDNA. Xma-3 did not cut rDNA extensively, indicating t,hat cytosincresidues are frequently methylated (result,s not shown). RamHT cleaves rDNA to fragments of about 5.3 and 3.6 kb (%ig. 1). Some full length (9.0 kb) repeat units remain, however; reflect,ing incomplete digestion, probably due to methylation (Gerlach bt Hedbrook, 1979). In the digest of ;MspT + KamHl (Fig. 2(a)), the largest) rlIN.4 fragments are around 3.0 kb but many smaller rDNA fragrnents exist. of differing lengt,h a,trd stoiohiometry, indica,tinp t,hat) t,heie are man? CCGG sites in rDNA but’ that~ in some repeat unitjs not all art’ (*leaved by ;WspI. The incompl& because t hr. c*xt,ernal digestion is presumably MspI + Xma-3

Lb!

‘!

:

h

i IW u TII

I Y I If I IYl 11’; I

<

, (b) A high proportion

i kb

(8 CpQ sequences arc in T-DNA but th,ere is some

methylated heterogeneity in methylated sites between grnes: one PCWG site is preferentially non-methylated

The enzymes HpaII, ,Wspl, X,ma-3 and HhaI do not cleave DNA when certain cytosine residues in their recognition sites are methylated. The locations of all the recognition sites for these enzymes in an rl)NA repeat unit have not been determined, but 39 HhaT, 15 Xma-3 and 19 HpaTT sites have been found by direct sequencing of 4.5 kb of the intergenic spacer region and the 3’ and 5’ ends of the 25 S and 18 S rRNA sequences of one gene (Barker et al., 1988). To investigate the extent of met)hylation at these sites t,hroughout, the rDNA

i 13 :

Figure 2. Distribution ancl rnrthglation ot’ (‘( ‘(Xi sites in rDh’A. I)h’A from “(‘hinese Spring” was digested with (a) MSJIT + KamHI or HJKIII + Krcn/ Hl and (h) HpaIT

+ EcoRT.

frac*tionated

I))

t~trc.troE)hor~sis.

with transferred to nitror~llulosr and hybridized 32P-labelrd rDh-A. Tn (c) DXi\t; f’ronr anruploids and Ej6IJTIii2 were restricted with NIRTlA E’coRI +Hhal. Ll and I,% refer t,o different ladders (SW the text). The sizes marked are in kb. (d) The lwatjion ot CCGG sites (1) in thr intrrgrnic spacer DiVu’,4 determined by direct sequencing of an isolat,ed repeat unit (Barker P[ al., 1988). The position of the preferentially now methylated (X(X: sequence in a subset of the genes is marked (1). T. Toy1 sit,rs: K. HrrmHT: E. E’coRl.

Regulation

of Cytosine

rytosine of some CCGG sequences is methylated. All the CCGG sites in rDNA have not been determined accurately but the 19 determined by direct DNA sequencing of part of one gene are shown in Figure 2(d). In the HpaII + BamHI digests (Fig. 2(a)) most of the rDNA exists in fragments characteristic of HamHI digests. Thus, most of the CCGG sites have the internal C methylated. EcoRI cleaves rDNA to repeat units of about 9.0 kb in length (Fig. 1). Much of the rDNA remains in 9.0 kb fragments after digestion with HpaII +EcoRI (Fig. 2(b)) or /!hnI+ EcoRT (Fig. 2(c)). Addition of more HpaII or HhaI or extra digestions does not change the hybridization patterns. These results also imply therefore that many of the CCGG and GCGC sites in the rDNA are methylated and in many rDNA repeat units all the CCGG and/or GCGC sites contain methylated cytosine residues. In the HpaII + BarnHI and HpaII +EcoRI digests, some ZIpaT cleavage products are in high stoiehiometry. The most prominent have lengths of 5.0, 3.5 and 1.8 kb in the HpaII +BumHT (Fig. 2(a)) and 2.7, 2.9 and 6.2 kb in the HpaII +EcoRT digests (Fig. 2(b)). These are due to cleavage of a large number of repeat units at one of the CCGG sites that lie close together in the intergenic spacer DNA, as marked in Figure 2(d). This position was confirmed by a series of digests involving EcoRI, TaqI, Hi&I or DdeI with HpaII. Fragments 2.7 and 2.9 kb in length are present in the HpaII + EcoRI digests because of the two major spacer length variants in this variety (see Fig. 1). There are more 2.7 kb fragments than 2.9 kb fragments, partly because there are more repeat units with shorter intergenic regions (from 1B and 6B NORs) than repeat units with longer intergenic regions from the 6B NOR (Fig. 1). In the EcoRI +HhaI digests (Fig. 2(c)) a ladder (I,I) of 12 hybridizing fragments is observed. Each fragment results from a cleaved GCGC site in only one of the array of 135 bp repeats that resides in t’he intergenic region (see Fig, 1). The ladder is due t)o different 135 bp repeats being cleaved in different genes. Whether this reflects variation in the distribution of GCGC sites or the pattern of methylation is not known unequivocally. However, HhaI sites are present in all or almost all repeats of the rRNA genes cloned (Appels & Dvorak, 1982; Barker et al., 1988) and rRNA genes within a locus are generally remarkably homogeneous in sequence (Barker et al.? 1988; Lassner & Dvorak, 1986). Furthermore, HhaI sites are present in the spacer repeats of genes at the 6B locus, as revealed in digests of DNA from plants lacking the 1B locus and in genes at) the 1B locus from digests of DNA lacking the 61~ locus (Fig. 2(c)). Therefore it is likely that the ladder of different fragments in Figure 2(c) reflects the methylation pattern. Another ladder of fragments, at lower concentration, labeled 1~2 in Figure 2(c), is visible after cleavage with HhaT. This ladder is due to multiple cleavages within the array of 135 bp repeats. At

Methylation

in rDNA

527

least ten fragment sizes make up this ladder (only 8 are visible in Fig. 2(c)), consistent with cleavages occurring within any of the 135 bp repeats. Other fragments occurring in high stoichiometry after cleavage with EcoRI and HhaI are approximately 4.0, 4.15 and 6-O kb. The 4.0 and 4.15 kb fragments probably result from cleavage at GCGC sites near the start of the 18 S rRNA sequence (Barker et al., 1988). There are many other rDNA fragments in lower stoichiometry in HpaII + BumHI, the HhaT + EcoRI and HpaII + EcoRI digests (Fig. 2(a), (b) and (c)). These are due to different CCGG or GCGC sites being cleaved in different repeat units. All these results imply that there is considerable heterogeneity within the rDNA family for the CCGG and GCGC methylation patterns. (c) Most genes unmethylated sites are &so unmethylated

at one or more CCGG at Some GCGC sites

In the above experiments it was shown that a fraction of the rRNA genes contains one or more unmethylated CCGG site(s) and there is also a fraction that contains some unmethylated GCGC sites. Are the two fractions of genes the same? This was investigated by carrying out single and double digestion with HpaII and HhaI after cleavage of DNA with EcoRI. The results (not shown) indicate that approximately half of the rRNA genes not cleaved by HpaII are cleaved by Nhal. Some of these genes are cleaved at HhaI sites in the 135 bp intergenic repeats. Thus, some genes that have all CCGG sites methylated do not have all GCGC sites methylated. This is not surprising, since there are many more HhaI than HpaII sites in each gene. However, almost all the genes cleaved by HpaII are also cleaved by HhuI and there appears to be few genes, if any, that are cleaved by IipaII but not HhaT. Thus, the genes that are not methylated at all their CCGG sites, including t’hose which are not methylated at the CCGG site in the intergenic spacer (Fig. 2(c)), also contain ma,ny unmethylated GCGC sites. (d) The cyltosine methyl&ion at C(,‘GG sites in a given rDNA locus is not invariant.. it depends on the number of rRNA genes in the cell

Cytological assays of nucleolar volume have suggested that the activity of an rDNA locus varies with the amount of rDNA in the cell (see Introduction). To investigate the cytosine methylation at CCGG sites in plants differing in the total number of rRNA genes per cell. DNAs were extracted from aneuploid lines that carry different numbers of NORs. These aneuploid derivatives of the variety “Chinese Spring” contained between approximately 3700 (nullisomic 6B t,etrasomic 6A) and 14,600 (tetrasomic 6B) rRNA genes per cell. Six equal portions of DNA were taken from each of two DNA preparations made from each genot,ype. Three portions were treated with IZpaIT and the other

R. B. I”lavell

528

Total

rRNA genes

et al.

x IO-‘/cell

5D+IA

Genes

m

‘B

c]

6B

in CS NORs

Figure 3. Kumber of rRPu’A genes cleaved by HpaII in plants containing different numbers of rRNA genes. The plants differing in total rDNA content are aneuploids containing the chromosomes 1H and 6R in different doses. These chromosomes carry blocks of rDNA as shown below. The numbers of genes in each NOR were taken from Flavell & O’Dell (1979). The number of genes cleaved by H&I are mean values of triplicate assays from 2 DR;A preparations. Portions of DNA were divided into 2 equal samples, incubated with HpaIJ or buffer then digest,ed wit)h EcoRT after adjustment of the salt concentration and then fractionated by electrophoresis, transferred to nitrocellulosr and hybridized with 32P-labelrd rDKA. The proportion of DPJA cleaved bv HpaIT was estimated by scanning suitable a;toradiograms. The number of genes cut was calculated assuming a valu; of 9100 for euploid “Chinese Spring” (Flavell & O’Dell, 1979).

three with buffer only. All six portions were then with EcoRI after raising the salt digested The DNA fragments were then concentration. fractionated by electrophoresis, transferred to nitrocellulose and hybridized with 32P-labeled cloned rDNA. The 9.0 kb hybridized fragments were estimated quantitatively by scanning suit’able autoradiograms. Preliminary experiments were carried out to ensure that the intensity of the signa,] on the autoradiogram was proportional to the DNA on the filter. The proportion of 9.0 kb rDNA repeat units cleaved by HpaII for each DNA was calculated from the areas under the scanned peaks for triplicate digests. The results, converted to numbers of genes, are shown in Figure 3. The number of genes cleaved by HpaTI is not) a constant percentage of the total. The deviation from a straight-line graph implies that as the number of genes per cell increases, the proportion of genes with all CCGG sites methylated increases. Plants with a total of 3700 rRNA genes have only 20 to 30% of their rDNA with all CCGG sites

methylated, while plants with 14,600 genes have 60 to 70% of their rRNA genes wit,h all (‘(XX sites methylat 15,000). the number having unmethylated (WG sites is unlikely to rise substantially above about’ 6500. Because the genetic location of t,he rRNA genes is known in these aneuploid plants, it is possible t,o consider the methylation status of specific rl)XA loci in different genetic backgrounds. The nullisomic2 6B tetrasomic 6A plant wit)h 3700 rRNA genes has most (72yb) of these genes in the 1H NORs. The tetrasomic 11~ plant’ with 11,700 rRNA genes differs from the euploid plant with 9100 genes in having an additional pair of the very same 1 H chromosom(as as those present in the nullisomic 6B tetrasomic 6.4 plant. However, only an additional 700 rRXA genc~ contain non-methylated (UK: sites in thts tetrasomic I 13 plants compared with the euploid plants, far fewer than the number caxptxc+cd (approximately 2000) if the 113 SOR methylation

Regulation of Cytosine

Methylation

in rDNA

529

6500 I !=4

.

%

x 5500 ~~ 5 ; 4500 2! x

. .

.

‘;; ;35001

I”

I 2500

. I.0

I.4

I,8

2.2

2.6

Ratto 2,7/2.9 : 6.2

Figure 4. The number of rRNA genes with all CCGG sites methylated in plants with different numbers of rRNA genes. The values plotted were calculated from the results shown in Fig. 3.

0

2

6

8

IO

12

14

Total no. of genes x lO-3

Figure 5. The ratio 2.7/2.9 : 6.2 kb rDNA fragments after digestion of different DNAs with HpaTT+EcoRI. The proportion of 2.7/2.9 to 6.2 kb rDNA were determined by scanning the autoradiograms obtained in the experiments

pattern were the same as in the nullisomic 6B tetrasomic 6A plants. Similar arguments apply to the 6B NOR genes when one compares the extent of methylation in the nullisomic 1B tetrasomic 1A plants with those in the euploid and tetrasomic 6B plants. Thus, the methylation status of CCGG sequences in an rDNA locus depends upon the number of rRNA genes in the genetic background. The more rRNA genes present elsewhere, the higher the proportion of genes at an NOR that have all CCGG sites methylated. Overall the results imply that the more rRNA genes are present the lower the probability that a gene will have one of its CCGG sites non-methylated. When the number of rRNA genes with all CCGG sites methylated is plotted against the total number of rRKA genes (Fig. 4), a graph is obtained that suggests that, even in plants with as few as 2000 rRNA genes, some of these genes are methylated at all CCGG sites. From the results in Figure 3, it was concluded that the average frequency of non-methylated sites per gene depends upon the total number of genes in the cell. Is bhis also true for the specific subfraction of genes that is preferentially non-methylated at the CCGG site in the intergenic spacer that produces 2.712.9 and 6.2 kb fragments upon treatment with HpaII and EcoRI? This question was investigated by scanning suitable autoradiograms and determining the relative proportions of rRNA gene equivalents present in 2.712.9 and 6.2 kb fragments in DNA for each aneuploid genotype. The ratio of rDNA in 2.7 + 2.9 fragments to that in 6.2 kb fragments is plotted against the total number of genes cleaved by HpaII in Figure 5. The latt’er axis was chosen because it represents the pool of genes with at least one CCGG sequence whose internal cytosine is non-methylated. For an rRNA gene that is cleaved only at the preferentially non-methylated CCGG site, the ratio of 2.7 or 2.9 to 6.2 kb should be approximately 0.45 (i.e. the ratio of the length of the fragments). All the ratios

4

described in Fig. 3.

measured were considerably higher than this, which shows that othel; cleavable sites in that gene fraction lie preferentially in the longer 6.2 kb fragments. The ratios decreased as the total number of genes with non-methylated cytosines per cell increased. This implies that: (1) in plants where fewer rRNA genes are present in this subfraction, the number of non-methylated CCGG sites per gene is higher, as also concluded for the total gene population from Figure 3; and (2) these additional sites lie preferentially in the 6.2 kb fragments, rather than in the 2.7j2.9 kb fragments. This latter finding is consistent with the fact that there are more HpaII sites in the 6.2 than in the 2.7 or 2.9 sites fragments (Fig. 2(d)) and non-methylated being distributed randomly between t,he 2.7, 2.9 and 6.2 kb fragments. (e) The rDNA in dominant, active NORs is less methylated than in less active NORs

After the discovery that the extent of methylation in CCGG sites of rDNA varied with the genetic background, it was desirable to find out whether specific sets of genes were, on average, less methylated than others, when in the same genetic background and whether the extent of methylation was related to gene expression or nucleolar the dominance. It was possible to investigate methylation status of specific gene sets where these could be distinguished using restriction endonucleases, as shown above for “Chinese Spring”. in this variety and some of its Furthermore, derivatives, the localization of the gene sets to specific NORs was known (see Fig. 1) and the relative activity of these NORs in terms of nucleolar volume had been measured (Table 1, Martini & Flavell, 1985). A correlation between reduced cytosine methylation

and enhanced

NOR

activity

was first

estab-

530

R. B. Flavell b

0

-HpoU

t

d

e

BumHI

+tiporl

. . Figure 6. Methyl&ion of CS and A. umbellulatu (KY) rDNA. DNAs from “Chinese Spring” (lanes b and c) and CS+ 1U (lanes d and e) were digested with HpaII + BumHI (lanes c and e) or BamHI (lanes b and d): fractionated by electrophoresis. transferred to nitrocellulose and hybridized with 32P-labeled rDNA.

Lane a, size marker DNAs of 4.4 and 3.5 kb. The DNA fragments produced when CS DNA is cleaved by HpaII at the preferentially non-methylated site are arrowed. A scan of lanes d and e of the autoradiogram is shown. The sizes are given in kb. The fragments from A. umbellulata and “Chinese Spring” are marked IT and CS. respectively. + Direction of electrophoresis.

lished by considering the results in Figure 3 and nucleolar volumes measured in these same aneuploid stocks (Martini & Flavell, 1985). When major NORs are deleted from euploid plants, the remaining NORs become more active to compensate for the loss (Table 1). Tn the nullisomic 6B plant the 1B nucleoli are twice as large as in euploid plants. In tetrasomic 1B plants the 1B nucleoli are smaller than in euploid plants (Martini & Flavell, 1985). As described above, the results in Figure 3 show that the degree of methylation of 1B rDNA in nullisomic 6B plants is less than in euploid and tetrasomic I B plants. Thus, decreased methylation is correlated with enhanced activity, The fact that the proportion of genes with all CCGG sites methylated increases as the total rDNA gene number increases is also consistent with this correlation. However, to gain more data on specific sets of genes, several additional experiments were

et al.

carried out on genotypes possessing NORs known to differ in activity. DNAs were extracted from CS euploid and also frotn substitut.ion or addition lines containing the NOR-bearing chromosome 1I’ from A umbellulata. Four derivative lines were examined. Threv possessed 1T,T substituted in place of wheat chromosomes 1A. 1H or 1D: respectively. while tht fourth contained a pair of 1V chromosomes in addition to the full wheat, complement. Most of the rRNA genes on chromosome lU havtb longer intergenic spacers (Martini et ~1.. 1982) than have any of the major classes of rRNA genes in “Chinese Spring”. The 1I.! genes can t.herefore be distinguished from the rRNA genes of “Chinese Spring” by restriction endonucleases. “(:hinese Spring“ and CSflU DNA digested with RamHT) HpaTI are shown in Figure 61 The BwmHT fragments of “Chinese Spring” are as shown in Figure 1. Those from the 1I’ NOR, t.hat include the intergenic spacer are about j.8 kb. Aft.er treatment with ZlpaIT, these 5.X kh fragments art‘ preferentially reduced relative to the (‘S 5.4 kh fragments (Fig. 6). Thus, a greater proportion of 1I‘ fragments cont8ain non-methylated CCGG sites compared with the CS fragments. The fragments normally produced by HpaIT digestion of CS rDNA (arrowed in Fig. 6) were not visible in CS+ 11’ HpaTI + BamHT digest.s, which suggested that t,he methylation of t,he CS rDNA at the special CC‘GG sit,e in the spacer had heen elevated in the presencc~ of the 1I: chromosome. This was confirmed more clearly 11)~ examining rDNA in EcoRI ) HpaII digests of CS. A. ~umbellulata rD?JA and t’he 1 I’ substitut,ion and addition lines (Fig. 7). A. umbellula.ta rI>NA contains several lengths of’ repeat unit, the predominant one is about 9.6 kh. i.e. longer than those of “Chinese Spring”, dutl to additional l)?c’A in the int,ergenic spacer (Fig, I). The CC(X site preferentially cleaved by IZpwTT in “(‘hinese Spring” is conserved in the 11.: r-DNA and so fragments of high st,oichiomctry are also observed in EcoRI + ?l;oaTT digests of ~1, umbell~ulatn TINA. These are 3.2, 2.95 and 2.75 kh and can be distinguished from bhose in “Chinese Spring” that are 2.9 and 2.7 kb (Fig. 7(a)). Aft.er digestion of the (1St 111 l)NAs with lr:~K,l and ff‘lpaT1, the tnajor fragmentas due t)o IIjuaIl cleavage are 3.2, 2.95 and 2.75 bp. i.rl. prcdominantly if not exclusively t’hosr from thr il. u.mbdbulatcz

rI)NA

(Fig. T(h)).

The

rDNA

on

chromosome 1I’ accounts for ahouf onlv %r)($, of’ the rJ)NA in t.he suhst,it.ution or addition hnc>,s (Martini rt al.. 1982) and thus the high stoichiomrtry of the Argilops 3.2 kh fragments rt~lativ~~ to the 2.7/2.9 kb wheat, fragments would not htt expect.ed if thrrr wertt no effect of the 1 I ’ chromosome on the wheat) rl)SA. Cf’tbconclude that t.he met,hyla,tion of the wheat, rl)XA at, t,hr spe(hia1 spa,cer site is cnhanc*ed in t.he prrsenc-tb of’ the 11’ chromosome. The 1IT NOR. is much more active than the ($ NORs

(Martini

cut nl..

1982).

Krc:~.usr~ its

prt~st*nc.~c’

Regulation of Cytosine Methylation A

in rDNA

531

B

Toq I

IB 66

-3.2 3.2

.Hpu!l

2.95 2.75

Acbo

(b)

(a)

Figure 7. Methyl&ion of CS and A. umbellulata NORs. (a) DNAs from A. umbellulata (lane a) and “Chinese Spring” (lane b) and (b), from “Chinese Spring” (lane a), CS 1U (1B) (lane b) and CS 1U (1D) (lane c) plants were fractionated by with HpaII + EcoRI, digested nitrocellulose and electrophoresis, transferred to 32P-labeled rDNA. The 3 major hybridized with A. umbellulata fragments and the 2 major CS fragments due to HpaII cleavage are arrowed in (a). The sizes of the fragments are in kb.

results in suppression of the CS NOR activity, it is said to be “dominant”. The results described here show therefore that the higher activity of the 1U NOR is associated with reduced cytosine methylation of its rDNA, while suppression of CS NOR with enhanced rDNA activity is correlated methylation. This relationship was further studied between wheat NORs by investigating the methylation of chromosome 1B versus 6B rDNA in CS euploid in which the 1B NOR is partially dominant to the 6B NOR. DNA was digested with TaqI) HpaII and the 1B and 6B gene fragments assayed by scanning autoradiograms after Southern hybridization with rDNA. The results of a typical scan are shown in Figure 8. A higher proportion of the 3.55 kb TaqI than of other TaqI fragments from IB genes is sensitive to HpaII. Thus, the 1B NOR of “Chinese Spring” contains a subset of genes that are considerably more sensitive to HpaII. Also, a greater proportion of the 3.1 kb TaqI fragments (NOR on 1B) is cleaved by HpaII than the 6B 2.6 and 2.8 kb TaqI fragments. If the 3.1, 2.8 and 2.6 kb fragments were cleaved by HpaII at the preferentially non-methylated site only, then the resulting fragments (arrowed in Fig. 8, lane A) should reflect the amounts of the 3.1, 2.8 and 2.6 kb cleaved. This is not found. There is an excess of the 3.1 kb cleavage product (Fig. 8). This implies that a higher proportion of the 2.8 and 2.6 kb Tap1 fragments are cleaved at sites other than

or

in

addition

to

the

preferentially

non-

+ h HPoII

. Figure 8. Methylation of different rRNA genes in CS wheat. CS DNA was divided into equal parts. One was incubated with HpaII (lane B), the other with buffer only (lane A). Both portions were then digested with TagI. After fractionation by electrophoresis and transfer to nitrocellulose, the DNA fragments were hybridized with 32P-labeled rDNA. Lane B shows the arrowed fragments produced when TaqI fragments a, b and c are cleaved by HpaII at the preferentially non-methylated site. The preferential cleavage of the highest molecular weight Tag1 fragment by HpaII is also illustrated. A representative scan of the Tap1 fragments a, b and c before and after HpaII cleavage is shown. The scan was taken from a shorter exposure of the autoradiogram than illustrated in lanes A and B.

methylated CCGG site. If in many of the 2.6 and 2.8 kb fragments there are additional non-methylated CCGG sites but only the special site in the 3.1 kb fragments, then the probability of cleavage of the 2.6 and 2.8 kb fragments by HpaII would be higher. This is not supported by the results in Figure 8 (scan before and after HpaII cleavage). Therefore, (1) a much greater proportion of the 3.1 kb fragments from 1B NOR must be nonmethylated at the special CCGG spacer site compared with the 2.8 and 2.6 kb fragments from the 6B NOR and (2) some of the 6B NOR genes must have a non-methylated CCGG other than at the special spacer site. There are twice as many 6B rRNA genes as IB rRNA genes and more 6B genes are cleaved by HpaII. However, it would appear that more genes at the 1B locus are cleaved at the

special site. The 1B NOR is partially

dominant over

532

R. B. Flavell

the 6B NOR even though it contains fewer genes (Table 1). Therefore, reduced cytosine methylation in CCGG sites is correlated with higher activity and the reduced CCGG methylation is predominantly at t,he special site in the intergenic spacer region (Fig. 1). This conclusion is also supported by the relative intensities of the 2.7 and 2.9 kb fragments after cleavage of CS DNA with EcoRI and HpaIT come from (Fig. 2(b)). The 2.7 kb fragments cleavage of the special spacer site in the 1B NOR genes and also in the 6B NOR genes with the shorter array of 135 bp repeats (see Fig. 1). The 2.9 kb fragments come from a similar cleavage in the 6B NOR genes with the longer array of 135 bp repeats. The ratio 2.7 : 2.9 fragments is greater than 2 which implies that there are more 1B NOR genes than 6B genes clea,ved at the special CCGG site.

et al.

The non-methylated CCGG sites, however, are not distributed at random, between all the rRNA genes. For example, in CS DNA a higher proportion of rRNA genes on chromosome 1B contain nonmethylated CCGG sites as compared to those on chromosome 6B. This is illustrated by the greater proportion of TaqI fragments being cleaved from the 1B genes upon treat,ment with HpaII (Fig. 8) and the higher concentrations of IB fragments than AB fragments that are cleaved at the preferentiall! non-methylated CCGG site (Figs 2(b) and 8). Also, a much higher proportion of non-methylated CCGG and GCGC sites are found in the 1U rDNA than in the 1B and 6B rDNA in plants containing all three chromosomes (Figs 6 and 7).

(1)) I’ytosine

,methylation

und

gene expression

4. Discussion (a) 7’he number of unmethylated in rDNA

cytosine residues is regulated

As expected from the high level of cytosine methylation in CpG dinucleotides and CXG trinucleot’ides in plant genomes (Gruenbaum et al., 1981), wheat rDNA repeat units were cleaved inefficiently by Xma-3, HpaII and HhaI. Since there were 15, 19 and 39 recognition sites for these enzymes respectively in only the intergenic DNA and the segments of the 18s and 25S sequences of the particular gene sequenced, the extent of CpG methylation is particularly high in wheat rDNA repeat units. The pattern of cytosine methylation in CCGG and GCGC sequences is complex. Many repeat units are resistant to HpaII and/or Hhal, indicating they are methylated at all the 19 CCGG and/or 39 GCGC sites determined by sequencing and at all the additional ones known to occur in t,he 18 S and 25 S gene sequences but not yet accurately mapped. In other rDNA repeat units, one or more CCGG or GCGC sites are not methylated, creating a heterogeneous cytosine methylation pat’tern within t,he multigene family (Fig. 2). However, one particular CCGG site is preferentially not methylated in a subset of t’he gene units and almost all these units also possess unmethylated GCGC sites. The proportion of rDNA repeat unit)s containing one or more non-methylated CCGG sites and the average number of non-methylat.ed CCGG sites within incompletely methylated repeat units is related to the number of rRNA genes in the cell (Figs 3, 4 and 5). As the number of rRNA genes increases (1) the proport’ion methylated at all CCGG sites increases (Fig. 3) and (2) amongst the genes that contain unmethylated CCGG sit,es, fewer genes contain more than one unmethylated CCGG site (Fig. 5). This relationship suggests t,he total number of unmethylated CCGG sites in rDNA might be relatively constant between genotypes. the sit’es being dist.ribut,ed among the available rDNA penes.

The distribution of non-methylat,ed CCGG sequences between different chromosomal loci correlates with the relative activities of the loci based upon nucleolar volumes (Table I). The most’ active locus has t)he highest proportion of genes wit.h non-methylated CCGG sequences and r*icr cersa. This was established by comparing the cytosine methylation of rDNA at LB and 6B loci in CS plants in which 1B loci are more active than 61% loci and at II’, 1B and 6B loci in plants of “Chinese Spring” containing 1IJ chromosomes from A. umbellulata, in which 1I’ loci are more active than 1K and 6B loci. Tt was also established bj finding that the proportion of genes with unmethylated CCGG sites at a locus decreased when t)he locus became more active, due to deletion of another rDNA locus (Fig. 3). This relationship is similar to that found for many other genes, viz. that activation of genes is associated with t,he loss of methyl groups .from specific cytosinr residues (Razin & Riggs, 1986: Disraeli & Szyf, 1984). In certain cases. the specific residues lit> a short distance upstream from where t ra,nsc*ription is initiated. The conserved and preferentially non methylated (KIGG site detected here in the rRNA genes of wheat is similarly located. Its position is about 164 base-pairs upstream from t’hr start ot transcription (Vincentjz & Flavrll. unpuhlishetl results) and downstream from t’he tandem array of 135 bp repeats. This region could be an importalit regulatory part of t,he promoter (Flavell P! cl/.. 1986). Hypomethylated sites in similar regions havt, also been described in subsets of the rKNA genes ot pea (Watson rt nl.. 1987) a.ntl flax (Blundy r’t tsl., 1987). Even when correlations ha,ve been rstahlishecl between hypornethylated regions in promoters antI gene expression. it IS oft’eri not possible to c~onclutl~~ whether demethytat,ion is thr primary (Avent in yrnc~ activation or results from gene activation (Hird. 1986). In the former case, one would postulate. for fat%ors t~ssent~ial for. f?xample. that important’

Regulation of Cytosine Methylation transcription cannot bind to the promoter if the cytosine residues are methylated and therefore the primary event) in gene activation must involve removing the methyl groups. In the latter case, one could postulate that genes that have been already activated have factors attached in regulatory regions that prevent methylation during DNA replication. Roth hypotheses can accommodate demethylation being essential for transcription. There is also the “hybrid” hypothesis in which the factors that bind to the promoter to convert a gene into an actJive state concurrently prevent methylation by chance, but demethylation is not essential for transcription, as concluded for Xenopus sperm rDNA when infected into oocytes (Macleod & Rird, 1983). To account for the non-random distribution of non-methylated CCGG sites between the rRNA genotypes, any factor genes in the different preventing methylation would have to have a greater affinity for, or be preferentially localized in, rRNA genes of lU> lB>6B. Tn the accompanying paper (Thompson & Flavell, 1988) we have shown that the promoter regions of some wheat rRNA genes possess sites sensitive to DNase T and the proportion of hypersensitive genes in a NOR is related t’o its activity. Also, the genes that carry hypersensitive DNase sit,es arc preferentially non-methylated at CCGG sites. Thus, the chromatin structure around the promoter of active rRNA genes is different from inactive genes and it is likely that this is associated with changes in cytosine methylation. We have speculated elsewhere (Flavell, 1986; Thompson & Flavell, 1988) that both altered chromatin structure and demethylation might be the result of genes being recruited into the active fraction by the binding of regulatory factors, in limiting concentration, t.o the regulatory regions of rRNA genes where the affinities for these factors are in t,he order lI:>lH>tiU.

On t,he assumption that highly methylated genes are not active, the results in this paper suggest that only a small proportion of rRNA genes of the total complement in wheat are act’ive. This is probably considerably fewer than 2000, even though most wheat genotypes possess between 8000 to 12,000 copies (Fig. 4). Thus, there is considerable opportunity for increasing the rates of rRNA synthesis by increasing t)he number of genes in the active fra,ction as well as. presumably, by increasing t’he number of t.ranscription complexes. The distribut’ion of non-methylated CCGG sites in a particular locus can change when it is in a different genetic background. For example, the extent of the methvlation of the 1B locus of rDNA in “Chinese Spring” is reduced in plants nullisomic for chromosome 6B but increased when in the pwsence of chromosome be predicted that as any a new genetic background its methylation pattern deJrending on its ability

1U. Consequently, it can rDNA locus is moved into in a breeding programme

may

change,

to compete

perhaps

for a limiting

in rDNA

533

factor. When do these changes occur’! Studies to be reported elsewhere by R. B. Flavell, R. A. Finch, and M. D. Bennett (unpublished results) suggest that restructuring of rDNA activity between loci starts in the fertilized egg cell but takes a few cell divisions before it is complete. Thus, it starts as soon as the new genotype is est’ablished. The necessity for a few cell divisions may reflect the need for DNA replication to occur before methylation and other patterns can be changed. Support for this work was provided by the Carnegie Institution of Washington, the AFRC and VSDA Competitive research grants 85CRCR,I- 1599 and 86CRCR-1-1910 to W.F.T.

References Appels, R. & Dvorak, ,J. (1982). Theoret. Appl. Genet. 63, 337-348. Barker. R. F., Harberd, N. P.. Jarvis, M. G. & Flavell. R. B. (1988). J. Mol. 61ioZ.201, l-18. Bird, A. (1984). Nature (London), 307, 503-504. Bird, A. (1986). Nature (London), 321, 209-213. Bird, A., Taggart,, M. M. & Gehring, C. A. (1981). J. Mol. Riol. 152, l-17. Blundy, K. S., Cullis, C. A. & Hepburn, A. G. (1987). Plant

Mol. Biol.

8, 217-225.

Ellis, T. N. H., Goldsbrough, P. B. & Cast.lrton, J. A. (1983). N,ILc~.Acids Res. 11, 3047-3064. Flavell, R. B. (1986). Oxford Surveys Plant Mol. Cell Biol. 3, 252-274. Flavell, R. B. & O’Dell, M. (1976). Heredity, 37, 377-385. Flavell, R. B. & O’Dell, M. (1979). C’hromosvmn (Berlin), 71: 135-152. Flavell, R. B.. O’Dell. M., Thompson, W. F.. Vincentz, M.. Sardana, R. & Barker, R. F. (1986). Phil. Trans. R. Sot., ser. B, 314, 385-397.

Gerlach, W. L. & Bedbrook, ,J. R. (1979). A+&. Acids Res. 7. 1869-1885. Gruenbaum, Y., Kaveh-Many, T., (“edar, M. bi Razin. A. (1981). ,Vature (London), 292. 86tb-862. La Volpe, A., Taggart, M.. MeStay, B. 8: Bird. A. (1983). Nucl. Acids Res. 11, 5361-5380. Lassner. M. & Dvorak, J. (1986). &~I. Acids Res. 14. 5499-5512.

Longwell. A. (‘. & Svihla, G. (1960). IL@. (‘rll Res. 20, 294.-312. Macleod. 1). & Bird. A. P. (1983). Xature (l&don), 306, 200.-203. Martini. G. & Flavell. R. B. (1985). Heredity, 54, Ill--120. Martini. (i., O‘Dell, M. & Flavell. R. B. (1982). (%rorrLosoma (&di?L) , 84, 687-700. Razin. A. & Riggs, A. D. (1980). Scivncr. 210, 604-610. Sears, E. R. (1954). Missouri Agrir. Eq. Station Res. Bull. 572. l-59. Southern, E. M. (1975). J. Mol. Biol. 98, 503.-517. Steele-Scott, N’.. Kavanagh, T. A. & Timtnis. *J. N. (1984). Pkmt Sci. Letters, 35. 213217. Thompson. W’. F. & Flavrll, R. B. (19x8). .I. Mol. Biol. 204, 535-548. Irchimiya. H.. Kato, H., Ohgawara, T.. Harada, H. & Sugiura. M. (1982). Plant Cell Physiol. 23. 1129.. 1131.

VVagner. 1. & Capesius. I. (1981). 654 . .52 I56.

Biochinr

Uiophys.

Acta,

534

R. B. E’lavell et al.

Waterhouse, R. N., Boulter, II. & Gatehouse, J. A. (1986). FEBS Letters, 209, 223-226. Watson, J. C., Kaufmann, L. S. & Thompson, W. F. (1987). J. MOE. Biol. 193, 15-26.

Yisraeli, J. $ Szyf: M. (1984). DNA Methylation Biochemistry and Biological Signi$cance (Razin R. 1 Cedar. H. & Riggs. A. II.. eds), pp. 353-378. Springer-Verlag, New York.

Edited by R. Laskey